Development and characterization of 29 polymorphic EST-SSR markers for Stipa purpurea (Poaceae).

PREMISE OF THE STUDY: Expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed using Illumina sequencing for further genetic diversity studies of Stipa purpurea (Poaceae). METHODS AND
RESULTS: Twenty-nine polymorphic and eight monomorphic EST-SSR loci were developed and characterized in 90 individuals from nine S. purpurea populations. The number of alleles per locus ranged from two to 13, and heterozygosity within populations and total heterozygosity ranged from 0.04-0.76 and from 0.04-0.87, respectively. Of 37 loci, 12 showed interspecific transferability and polymorphism in a related species, S. glareosa.
CONCLUSIONS: These newly developed EST-SSR primers provide a useful tool to investigate genetic diversity at the population level and to analyze the population structure of S. purpurea.

Stipa purpurea Griseb. (Poaceae) is widely distributed along the precipitation gradient from the southeast to the northwest of the Qinghai–Tibet Plateau (Yue et al., 2011). It is a species endemic to the Qinghai–Tibet Plateau, and it plays a prominent role in protecting the ecological environment by acting as a windbreak, fixing sand, conserving water and soil, and preventing grassland degradation (Yue et al., 2008). Stipa purpurea is also important for the development of animal husbandry because of its high nutritional value and good palatability (Yue et al., 2008), but it has suffered natural and anthropogenic disturbances in recent years. Most studies on S. purpurea have focused on its biological characteristics (Li et al., 2015); however, little is known about its population genetic diversity (Yue and Peng, 2014).

To our knowledge, only a limited number of simple sequence repeat (SSR) markers (using the Fast Isolation by AFLP of Sequences COntaining repeats [FIASCO] protocol) (n = 15; Liu et al., 2011) and intersimple sequence repeat (ISSR) markers (n = 8; Liu et al., 2009) have been developed for use in S. purpurea to investigate its genetic diversity in different populations (Liu et al., 2009, 2011; Zhai, 2012). Furthermore, the development of microsatellite markers in S. purpurea has been very slow due to the lack of genome sequences. Thus, developing a greater number of microsatellite loci for this species is necessary for population genetic diversity studies of S. purpurea to progress. Next-generation sequencing allows for rapid development of a large number of SSR markers (Huang et al., 2014). Recently, we sequenced the S. purpurea transcriptome using the Illumina next-generation sequencing platform to understand drought tolerance (Yang et al., 2015). Here, we report the rapid and cost-effective development of 29 novel polymorphic expressed sequence tag (EST)–SSR markers for S. purpurea, which will be useful in future studies of population genetics in this species.

METHODS AND RESULTS

Transcriptome sequencing of S. purpurea was conducted using an Illumina Genome Analyzer (Illumina, San Diego, California, USA). Approximately 51 million 75-bp paired-end reads were obtained and assembled into 84,298 unigenes with mean sizes of 579 nucleotides (Yang et al., 2015). SSRs were detected using the MIcroSAtellite Identification Tool (MISA; Thiel et al., 2003), with the criteria of eight, five, five, five, and five repeat units for di-, tri-, tetra-, penta-, and hexanucleotide motifs, respectively. A total of 2105 SSRs were identified, with trinucleotide repeats (98.9%, 2081) being the most common, followed by dinucleotide (0.8%, 17), hexanucleotide (0.2%, 4), and tetranucleotide (0.1%, 3) repeats. Primer Premier 5 software (PREMIER Biosoft International, Palo Alto, California, USA) was used to design 50 primer pairs (GenBank accession numbers: KP729144–KP729174, KU987914–KU987932), 18–21 bp long, that amplified product sizes ranging from 100–600 bp.

Polymorphisms of these primer sets were assessed in 90 S. purpurea individuals from nine populations (10 individuals per population) in Tibet (Fig. 1, Appendix 1). We also chose S. glareosa P. A. Smirn. (Poaceae) (10 individuals; 32°39′47″N, 79°55′48″E) to test the cross-species amplification of polymorphic and monomorphic markers in S. purpurea. Voucher specimens (Appendix 1) were deposited in the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN).

Fig. 1.

Sampling locations of Stipa purpurea and S. glareosa in Tibet.

Genomic DNA was extracted from leaf tissues using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). PCR amplifications were performed in 20-μL reaction mixtures containing 0.5 units of Taq polymerase (TaKaRa Biotechnology Co., Dalian, China), 2 μL 10× PCR buffer (200 mM Tris-HCl [pH 8.8], 100 mM (NH4)2SO4, 100 mM KCl, 1% Triton X-100, 20 mM MgSO4), 1.6 μL dNTPs (2.5 mM each), 0.5 μL each primer (10 μM), and 1 μL of genomic DNA (∼50 ng/μL). PCR conditions comprised an initial denaturing step at 94°C for 4 min; followed by 35 cycles of 94°C for 30 s, appropriate annealing temperatures (Table 1) for 30 s, and 72°C for 35 s; and a final extension at 72°C for 5 min. PCR products were first detected using 1.0% agarose gel electrophoresis, then run on 6% denaturing polyacrylamide gels and silver stained. The band size was calculated by comparison with a 25-bp DNA ladder (Fermentas, Vilnius, Lithuania; Yan et al., 2011). Stipa purpurea is a tetraploid species (2n = 4x = 44) (Sheidai and Attaei, 2005), so traditional measures of genetic variability such as deviation from linkage disequilibrium and Hardy–Weinberg equilibrium (HWE) could not be determined. However, GenoDive v.2.0b20 software (Meirmans and Van Tienderen, 2004) enables the analysis of amplified polymorphic fragments (bands) from polyploids.

Table 1.

Characteristics of 37 EST-SSR primers developed for use in Stipa purpurea.

Locus	Primer sequences (5′–3′)	Repeat motif	Allele size range (bp)	Ta (°C)	GenBank accession no.
ZH1	F: GCGGAACAAGGAGAACC	(CGC)8	252–269	53	KP729144
	R: ATAGACATTTCCGCCAGC
ZH2	F: CGCCATCGCTCGCAGTA	(CCG)6	45–65	53	KP729145
	R: TACGACTGGAAGGGCGAG
ZH3	F: AGCCAGACGACCAAGAACC	(CCG)6	357–374	53	KP729146
	R: TACAGCGACAACGACATGGAC
ZH4	F: CAAGACCAAGCCAGTA	(GAA)7	230–250	53	KP729147
	R: AAAACGGGAACTGTGA
ZH5	F: GCGAGTTCTGGCAGTTCA	(CGG)4	379–390	50	KP729148
	R: CAAGTCCATCGGGAGGC
ZH6	F: AGACGATTGGGTGCTGTGC	(AAG)3	57–81	53	KP729149
	R: GCGAAGAGCGACGAGTAG
ZH7	F: GCGTCCAACTCCAAGAA	(GTG)8	43–66	52	KP729150
	R: ACGAGGCAAAGAGTCCA
ZH8	F: CCAGCACCGCCGATGTA	(CCA)8	71–94	53	KP729151
	R: TTGGACAGGCTGAGTAGG
ZH9	F: TGAAGAAGACCACCATCGC	(CAG)8	153–176	53	KP729152
	R: CCTCCACAGCGTGTAATCC
ZH10	F: CCTCCTCGCAGTTCTTCC	(CCT)5	125	47	KP729153
	R: CCACCTGTTCCCATCCTC
ZH11	F: CGCCAATAACTGCGGCTTC	(CCT)2	342–348	51	KP729154
	R: CGGCGAGGAGGAATCAGAGG
ZH12	F: TCCCCAGACTCCAATCCTTCC	(AGC)7	191–211	51	KP729155
	R: TCAATTGCGGGCTCATTGC
ZH13	F: TAGCATCAGCGGCACCTC	(CCG)6	70–87	51	KP729156
	R: GCGGCTTGCTTGTTTCCT
ZH14	F: CCTCCAGTGAGCAACCCA	(TGC)4	193–204	50	KP729157
	R: GACGGCAGACGACTCCTT
ZH15	F: AGGCTCAGCAGCAAAGA	(CCA)5	456–464	50	KP729158
	R: CAGAAGTGGACGCAAAC
ZH16	F: GGTGAAGGAGGGTTGCG	(CCA)2	303–308	52	KP729159
	R: TGTCGGTGCCGTTGCTG
ZH17	F: CCAAAAACCAAGCGAACCGA	(CCG)6	252–269	51	KP729160
	R: TTTGTTGGCCTCATCCTCGT
ZH18	F: ACACTCCCAGTTCAGCCATC	(CAG)7	542–562	51	KP729161
	R: CGTGGTACCATCTGGCCTTG
ZH19	F: CTGTGGCTACTCGTGAT	(CAG)7	416	53	KP729162
	R: CGATAAAGGCAGATAGTAAA
ZH20	F: CCCACTTCGGCGGCATCAT	(CAG)6	380–397	51	KP729163
	R: AGCATCGGTCGCAGGGAGGA
ZH21	F: AGGCTCCATCCATCTTTACT	(TGC)6	42–59	50	KP729164
	R: TTTCAGATAACCACCAGATT
ZH22	F: CCCTCATCGCCATCTTTG	(AGC)4	161–172	50	KP729165
	R: GCACTCCTGCCACTCCAT
ZH23	F: GCATCCATCCCTACCTCA	(AAG)7	198–218	50	KP729166
	R: CAGCGTCACCATTAGCAG
ZH24	F: GCTGCTCCTCATCGTCGTCT	(CCT)4	80–91	51	KP729167
	R: GCCTTCACCTTCTTGCCCT
ZH25	F: CTCGCGTGATTTCCAAACCC	(CCG)2	484–489	51	KP729168
	R: CGCAACCCTAGCTAACAACA
ZH26	F: TCATCAAGCTCTTCCTGCCG	(CCG)3	50–58	51	KP729169
	R: GCCGCCATTTCCATTTCCAT
ZH27	F: GCGGATGAGGAAGTAGAGG	(AAG)2	53–58	51	KP729170
	R: GCAGAAGGTCCATCAACA
ZH28	F: GCTCCCTACCGTCCTCCTC	(CT)13	53–78	55	KP729171
	R: TGGGTTGGGTGGTGGCTC
ZH29	F: CGGCGAGCGAACTGTCCAT	(AAC)5	172–186	55	KP729172
	R: CGCTTGAGGGTAGCCAGATGA
ZH30	F: AGCACTTGGCAACCTGAA	(GA)5	571–580	53	KP729173
	R: GCATCAAGCCTCACAAACCAT
ZH31	F: TACTGCCATTGCCACCTT	(AGC)8	515–538	50	KP729174
	R: TCGCCGCATTCGTTGT
ZH32	F: GTCGCCGCATTGTCATCAG	(CCA)6	282	53	KU987914
	R: TATCGGTGCTGGAGGAGGC
ZH33	F: GCCCAGTTCTTGGCTATCTTAC	(CAC)5	282	53	KU987915
	R: CTGCTGAGAAACGGTGGGT
ZH34	F: CAACTCGCTGGTATCGTGC	(CAC)7	154	55	KU987916
	R: TCTCGGCTATGTCAGGTGC
ZH35	F: ACCGTGGCACAGGCTCCGTC	(GCA)7	507	55	KU987917
	R: AGGCATCCCATTGCGTAG
ZH36	F: CACAAGGGTTACTGGGATA	(CCA)6	291	55	KU987918
	R: AACGGTGGGTGCGGATG
ZH37	F: TGGCACAGGCTCCGTC	(GCA)7	435	53	KU987919
	R: TCCCATTGCGTAGGTAG

Note: Ta = annealing temperature.

Of the 50 primer pairs, 29 (58%) amplified a polymorphism after excluding those that did not amplify (13, 26%) or were monomorphic (8, 16%) (Table 1), and this was assessed using 90 individuals from nine populations. The 29 polymorphic SSR markers were analyzed using GenoDive v.2.0b20 software, and the number of alleles per locus (A) according to Nei (1987) ranged from two to 13 alleles with a mean of 4.07 alleles per locus. The average heterozygosity within populations (Hs) and total heterozygosity (Ht) ranged from 0.044 to 0.756 and 0.044 to 0.868 with averages of 0.38 and 0.43, respectively, suggesting that genetic diversity was higher within than among populations. HWE (P < 0.05) tests showed that no loci significantly deviated from the equilibrium between locus pairs, suggesting that the nine populations investigated were in genetic equilibrium. All loci in the current study, including 29 polymorphic and eight monomorphic primers in S. purpurea, were also screened in cross-amplification tests of 10 S. glareosa individuals. Twelve of the 37 primers were successfully amplified and all revealed polymorphisms. A varied from two to three, and Hs and Ht ranged from 0.50 to 0.83 and 0.50 to 0.83 with averages of 0.58 and 0.58, respectively (Table 2).

Table 2.

Results of initial primer screening for the 29 polymorphic loci in Stipa purpurea and cross-species amplification in S. glareosa.

	Stipa purpurea																														Stipa glareosa
	Nagqu (N = 10)			Bangor (N = 10)			Nyima (N = 10)			Tsochen (N = 10)			Tradom (N = 10)			Burang (N = 10)			Menshi (N = 10)			Zanda (N = 10)			Shiquanhe (N = 10)			Total (N = 90)			Shiquanhe (N = 10)
Locus	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht	A	Hs	Ht
ZH1	2	0.533	0.533	2	0.356	0.356	2	0.533	0.533	2	0.356	0.356	2	0.533	0.533	2	0.356	0.356	2	0.356	0.356	2	0.533	0.533	2	0.356	0.356	3	0.444	0.451	—	—	—
ZH2	2	0.356	0.356	3	0.622	0.622	3	0.622	0.622	2	0.533	0.533	1	0.000	0.000	2	0.356	0.356	2	0.533	0.533	2	0.356	0.356	2	0.356	0.356	3	0.489	0.621	—	—	—
ZH3	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	2	0.000	0.000	1	0.000	0.000	2	0.044	0.044	2	0.500	0.500
ZH4	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.533	0.533	1	0.000	0.000	2	0.044	0.232	—	—	—
ZH5	3	0.622	0.622	1	0.000	0.000	2	0.533	0.533	2	0.533	0.533	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	2	0.711	0.711	2	0.356	0.356	3	0.456	0.531	2	0.667	0.667
ZH6	2	0.533	0.533	2	0.356	0.356	3	0.711	0.711	3	0.622	0.622	3	0.711	0.711	2	0.356	0.356	4	0.800	0.800	3	0.571	0.571	2	0.356	0.356	4	0.644	0.644	—	—	—
ZH7	4	0.800	0.800	3	0.622	0.622	2	0.533	0.533	5	0.889	0.889	3	0.711	0.711	2	0.356	0.356	4	0.800	0.800	2	0.711	0.711	4	0.800	0.800	13	0.756	0.868	3	0.833	0.833
ZH8	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	3	0.622	0.622	2	0.356	0.356	2	0.356	0.356	1	0.000	0.000	3	0.356	0.356	3	0.622	0.622	5	0.333	0.356	—	—	—
ZH9	2	0.356	0.356	1	0.000	0.000	2	0.533	0.533	3	0.711	0.711	2	0.356	0.356	2	0.356	0.356	2	0.533	0.533	2	0.000	0.000	2	0.356	0.356	4	0.444	0.572	2	0.500	0.500
ZH11	2	0.356	0.356	2	0.533	0.533	1	0.000	0.000	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	1	0.533	0.533	1	0.000	0.000	5	0.378	0.473	—	—	—
ZH12	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	3	0.714	0.714	1	0.000	0.000	2	0.356	0.356	2	0.533	0.533	2	0.000	0.000	3	0.622	0.622	4	0.222	0.222	—	—	—
ZH13	1	0.000	0.000	2	0.356	0.356	2	0.356	0.356	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	2	0.356	0.356	1	0.356	0.356	2	0.356	0.356	4	0.178	0.178	2	0.500	0.500
ZH14	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	1	0.000	0.000	2	0.356	0.356	2	0.356	0.356	1	0.000	0.000	5	0.600	0.588	2	0.667	0.667
ZH15	3	0.622	0.622	2	0.356	0.356	2	0.533	0.533	3	0.622	0.622	3	0.711	0.711	3	0.711	0.711	2	0.533	0.533	2	0.000	0.000	2	0.356	0.356	2	0.222	0.202	—	—	—
ZH16	1	0.000	0.000	1	0.000	0.000	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	1	0.000	0.000	1	0.356	0.356	2	0.356	0.356	3	0.467	0.469	2	0.500	0.500
ZH17	2	0.533	0.533	2	0.356	0.356	2	0.533	0.533	2	0.356	0.356	2	0.533	0.533	2	0.356	0.356	2	0.356	0.356	2	0.800	0.800	2	0.356	0.356	6	0.489	0.635	2	0.667	0.667
ZH18	2	0.533	0.533	3	0.711	0.711	3	0.711	0.711	1	0.000	0.000	3	0.622	0.622	1	0.000	0.000	1	0.000	0.000	4	0.533	0.533	3	0.711	0.711	4	0.189	0.21	2	0.500	0.500
ZH20	4	0.857	0.857	2	0.667	0.667	3	0.622	0.622	2	0.356	0.356	2	0.429	0.429	3	0.714	0.714	2	0.533	0.533	2	0.711	0.711	1	0.000	0.000	5	0.756	0.77	—	—	—
ZH21	4	0.8	0.8	3	0.622	0.622	4	0.800	0.800	3	0.622	0.622	3	0.711	0.711	3	0.622	0.622	3	0.622	0.622	3	0.000	0.000	2	0.533	0.533	4	0.211	0.211	—	—	—
ZH22	1	0.000	0.000	2	0.356	0.356	3	0.622	0.622	1	0.000	0.000	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	1	0.356	0.356	2	0.356	0.356	2	0.178	0.267	2	0.500	0.500
ZH23	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	2	0.356	0.356	2	0.356	0.356	1	0.000	0.000	2	0.000	0.000	2	0.356	0.356	5	0.389	0.39	2	0.500	0.500
ZH24	2	0.533	0.533	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	3	0.622	0.622	1	0.000	0.000	1	0.000	0.000	2	0.533	0.533	2	0.111	0.127	—	—	—
ZH25	2	0.429	0.429	2	0.356	0.356	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.533	0.533	1	0.000	0.000	3	0.456	0.487	2	0.596	0.596
ZH26	1	0.000	0.000	1	0.000	0.000	2	0.571	0.571	2	0.356	0.356	2	0.533	0.533	3	0.622	0.622	2	0.429	0.429	2	0.356	0.356	3	0.711	0.711	2	0.044	0.044	—	—	—
ZH27	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	1	0.000	0.000	2	0.356	0.356	1	0.000	0.000	9	0.633	0.794	—	—	—
ZH28	2	0.356	0.356	4	0.800	0.800	3	0.711	0.711	4	0.800	0.800	2	0.356	0.356	1	0.000	0.000	5	0.889	0.889	2	0.533	0.533	4	0.800	0.800	3	0.478	0.555	—	—	—
ZH29	1	0.000	0.000	2	0.533	0.533	3	0.622	0.622	2	0.533	0.533	3	0.711	0.711	1	0.000	0.000	2	0.533	0.533	2	0.714	0.714	2	0.356	0.356	5	0.733	0.733	—	—	—
ZH30	2	0.571	0.571	3	0.622	0.622	3	0.800	0.800	3	0.622	0.622	2	0.533	0.533	3	0.711	0.711	3	0.800	0.800	3	0.000	0.000	4	0.800	0.800	3	0.289	0.453	—	—	—
ZH31	1	0.000	0.000	2	0.533	0.533	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	2	0.356	0.356	1	0.356	0.356	1	0.000	0.000	3	0.289	0.453	—	—	—

Note: A = number of alleles; Hs = heterozygosity within populations; Ht = total heterozygosity.

CONCLUSIONS

This is the first known report of EST-SSRs developed for use in S. purpurea. Twenty-nine polymorphic and eight monomorphic primer sequences were described in this study. The markers described here appear to be highly reliable and will enable the investigation of genetic diversity at the population level and the analysis of population structure of S. purpurea. They may also contribute to studies of genetic diversity across other Stipa species.

Appendix 1.

Locality and accession information of the nine Stipa purpurea populations and a single S. glareosa population of the Qinghai–Tibet Plateau used in this study. All specimens are deposited in the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN), Kunming, Yunnan, China.

Population code/Species	Collection locality	Voucher specimen accession no.	Altitude (m a.s.l.)	Geographic coordinates	N
P1/S. purpurea Griseb.	Nagqu, Tibet	1270416	4690	30°50′52″N, 90°37′15″E	10
P2/S. purpurea	Bangor, Tibet	1270417	4636	31°36′58″N, 89°32′14″E	10
P3/S. purpurea	Nyima, Tibet	1270411	4538	32°00′05″N, 86°50′54″E	10
P4/S. purpurea	Tsochen, Tibet	1270414	4794	31°03′06″N, 85°05′42″E	10
P5/S. purpurea	Tradom, Tibet	1270412	4651	30°14′54″N, 82°58′31″E	10
P6/S. purpurea	Burang, Tibet	1270415	4639	30°42′57″N, 81°21′39″E	10
P7/S. purpurea	Menshi, Tibet	1270408	4495	31°10′57″N, 80°47′22″E	10
P8/S. purpurea	Zanda, Tibet	1270409, 1270410	4584	31°31′43″N, 79°58′42″E	10
P9/S. purpurea	Shiquanhe, Tibet	1270406	4682	32°39′47″N, 79°55′48″E	10
P9/S. glareosa P. A. Smirn.	Shiquanhe, Tibet	1270424	4682	32°39′47″N, 79°55′48″E	10

Note: a.s.l. = above sea level; N = number of individuals.